Method of regenerating connective tissue with a scaffold of coral-derived collagen

ABSTRACT

A method is disclosed for regenerating connective tissue by administering a scaffold comprising collagen fibers extracted from a soft coral. The length of the soft coral collagen fibers following stretching by about 15% is identical to the length of the fibers prior to stretching.

RELATED APPLICATIONS

This Application is a division of U.S. patent application Ser. No.12/934,704 filed on Oct. 28, 2010, which is a National Phase of PCTPatent Application No. PCT/IL2009/000334 having International filingdate of Mar. 25, 2009, which claims the benefit of priority of U.S.Provisional Patent Application No. 61/064,792 filed on Mar. 27, 2008.The contents of the above Applications are all incorporated by referenceas if fully set forth herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to acollagen comprising enhanced elasticity and, more particularly, but notexclusively, to a coral-derived collagen.

Collagens are the main structural proteins responsible for thestructural integrity of vertebrates and many other multicellularorganisms.

Collagen provides biomaterials for a myriad of uses includingpharmaceutical (haemostatic compresses, sponges, dressings in particularhealing dressings), medical (prostheses such as cardiac valves, tendonsand ligaments, skin substitutes, filling agents), odontological (gumimplants) and cosmetic (additive, anti-wrinkling agent, microcontainerfor perfumed substances). Collagen-based products can be made intomembranes, films, sheets, sponges and dispersions of fibrils for any ofthe above purposes.

One important area in which collagen has proven useful is that of tissueengineering. Tissue engineering (TE) is defined as the application ofengineering disciplines to either maintain existing tissue structures orto enable new tissue growth. This engineering approach generallyincludes the delivery of a tissue scaffold that serves as anarchitectural support onto which cells may attach, proliferate, andsynthesize new tissue to repair a wound or defect. Tissue scaffoldstypically have high open-celled porosity to allow cell migrationthroughout the scaffold and also to allow important nutrient-bearingfluids to flow through the scaffold to maintain the health of the cells.

Tissue engineering scaffolds that have been reported in the literatureinclude meshes, woven structures, non-woven structures, knittedstructures, three dimensional woven structures, sponges and foams. Thescaffolds are typically made of materials that are biocompatible. Often,they are made of biodegradable materials. Biodegradable materialsreadily break down into small segments when exposed to moist bodytissue. The segments then either are absorbed by the body, or passed bythe body. More particularly, the biodegraded segments do not elicitpermanent chronic foreign body reaction, because they are absorbed bythe body or passed from the body, such that no permanent trace orresidual of the segment is retained by the body. Ideally, thebiodegradable tissue scaffolds degrade at approximately the same rate asthe body synthesizes new tissue to repair the wound or defect.

A broad range of tissue engineering products based on collagen scaffoldsare currently under development, and some of them have already reachedthe market. For example, collagen gels seeded with fibroblasts have beenused as the “dermal” layer of the artificial skin sold under thetradename APLIGRAFT (Sandoz A G, Basel, Switzerland), and collagensponges have been used as an osteoconductive carrier of bone morphogenicprotein-2 (BMP-2) for spine fusion and the treatment of long bonefractures.

Collagen based biomaterials have been formed into fibers, film, sheets,sponges and dispersions of fibrils. Many of these forms could be used astissue engineering scaffolds in the repair or augmentation of bodytissue.

RELATED ART

U.S. Pat. No. 20050271614 teaches use of collagen of aquatic origin forcosmetic, pharmacological, dental, and cell culture products.

U.S. Pat. No. 20060210601 teaches a processed (non-native) collagen ofenhanced elasticity and mechanical endurability.

Summary of the Invention

According to an aspect of some embodiments of the present inventionthere is provided an isolated collagen fiber, wherein a length of thefiber prior to stretching by about 15%, is identical to a length of thefiber following the stretching by about 15%.

According to an aspect of some embodiments of the present inventionthere is provided an isolated collagen fiber, comprising a NuclearMagnetic Resonance (NMR) spectroscopic profile as shown in FIG. 1.

According to an aspect of some embodiments of the present inventionthere is provided an isolated collagen fiber being extracted from a softcoral Sarcophyton sp.

According to an aspect of some embodiments of the present inventionthere is provided a scaffold comprising the collagen of the presentinvention.

According to an aspect of some embodiments of the present inventionthere is provided a cell culture comprising mammalian cells seeded onthe collagen of the present invention.

According to an aspect of some embodiments of the present inventionthere is provided a composite comprising, as a first component, thecollagen fiber of the present invention and a second component selectedfrom the group consisting of a mineral, a polysaccharide and apolypeptide.

According to an aspect of some embodiments of the present inventionthere is provided a method of regenerating tissue, the method comprisingproviding to a subject in need-thereof the scaffold of the presentinvention, thereby regenerating tissue.

According to an aspect of some embodiments of the present inventionthere is provided a method of farming a soft coral, the methodcomprising: (a) attaching the soft coral to a clay surface and (b)growing the soft coral on the clay surface under conditions whichsupport propagation, thereby farming the soft coral.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the isolatedcollagen fiber of the present invention.

According to an aspect of some embodiments of the present inventionthere is provided a cosmetic composition comprising the isolatedcollagen fiber of the present invention.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polypeptide comprising a Nuclear MagneticResonance (NMR) spectroscopic profile as shown in FIG. 1.

According to some embodiments of the invention, the isolated collagenfiber has an amino acid composition as shown in FIG. 2B.

According to some embodiments of the invention, the isolated collagenfiber is extracted from a coral.

According to some embodiments of the invention, the coral is Sarcophytonsp.

According to some embodiments of the invention, a fragment of thecollagen comprises a major mass at about 4118.47 mass unit (MU).

According to some embodiments of the invention, the isolated collagenfiber comprises a Mass spectroscopy (MS) profile as shown in any one ofFIGS. 13A-B-16A-B.

According to some embodiments of the invention, the isolated collagenfiber comprises a stiffness about 30-50% lower than that of mammaliancollagen.

According to some embodiments of the invention, the isolated collagenfiber comprises a stiffness between about 0.34 GPa and 0.54 GPa.

According to some embodiments of the invention, the isolated collagenfiber comprises a tensile strength about half of mammalian collagen.

According to some embodiments of the invention, the isolated collagenfiber comprises a tensile strength between about 39-59 MPa.

According to some embodiments of the invention, each polypeptide of atriple helix of the collagen is separated by a 100 nm spacing.

According to some embodiments of the invention, the isolated collagenfiber is resistant to degradation by trypsin and collagenase.

According to some embodiments of the invention, the scaffold comprises(i)

a support; and (ii) a layer being attached to at least part of a surfaceof the support, the layer comprising the collagen of the presentinvention.

According to some embodiments of the invention, the scaffold furthercomprising cells.

According to some embodiments of the invention, the conditions comprisea water temperature at a range of about 20-26° C. under a lightintensity range of about 35-130 μE.

According to some embodiments of the invention, the conditions comprisea pH of about 8.2.

According to some embodiments of the invention, when the temperature isabout 20° C., the light intensity is about 230 μE.

According to some embodiments of the invention, when the temperature isabout 26° C., the light intensity is about 250 μE.

According to some embodiments of the invention, the method furthercomprises cutting the soft coral into pieces of less than 50 mm² andgreater than 25 mm² prior to the attaching.

According to some embodiments of the invention, the attaching iseffected using a glue.

According to some embodiments of the invention, the soft coral is of theSarcophyton genus.

According to some embodiments of the invention, the soft coral comprisesSarcophyton sp or Sarcophyton glaucum.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a table illustrating the results of proton and carbon NMRanalysis of the collagen of the present invention.

FIGS. 2A-B illustrate the results of amino acid analysis of the collagenof the present invention.

FIG. 3 is an image of isolated collagen. Fibrils are clearly presentrunning from the lower left of the frame to the upper right of theframe. A “*” marks a fibril with ˜100 nm spacing.

FIGS. 4A-B are image from small fixed samples of the collagen of thepresent invention. No fibrillar organization is present at the 15 μmscale isolated collagen (FIG. 4A) or at the 1 μm scale (FIG. 4B).

FIG. 5 is an image from a large unfixed sample of the collagen of thepresent invention. Probe placement was difficult and no fibrillarorganization was present at the 3 μm scale.

FIG. 6 is a Raman spectrum obtained from an isolated collagen sample.

FIG. 7 is an example of image data from a collagen sample of the presentinvention.

FIG. 8 is a graph illustrating raw force-time data for a collagen sampleof the present invention.

FIG. 9 is graph illustrating force displacement data for a collagensample of the present invention.

FIG. 10 is a stress-strain curve obtained from force-displacement data.

FIG. 11 is a table illustrating amino acid normalization results for asample of the collagen of the present invention.

FIG. 12 is a table illustrating amino acid normalization results for asample of the collagen of the present invention.

FIGS. 13A-B is a read-out of MALDI MS analysis of 1 pmol of sample 1 ofthe collagen of the present invention.

FIGS. 14A-B is a read-out of MALDI MS analysis of 1 pmol of sample 1 ofthe collagen of the present invention.

FIGS. 15A-B is a read-out of MALDI MS analysis of 1 pmol of sample 2 ofthe collagen of the present invention.

FIGS. 16A-B is a read-out of MALDI MS analysis of 1 pmol of sample 2 ofthe collagen of the present invention.

FIG. 17 is a diagram of the loading profile of an experiment describedherein.

FIG. 18 is a stress-strain curve of the full experiment, loading profileA.

FIG. 19 is a stress-strain curve of the final cycle, loading profile A.

FIG. 20 is a derivative of the stress-strain curve of the last cycle,loading profile A.

FIG. 21 is a stress-strain curve of the full experiment, loading profileB.

FIG. 22 is a stress-strain curve of the stretch at the first cycle,loading profile B.

FIG. 23 is a derivative of the stress-strain curve of the stretch at thefirst cycle, loading profile B.

FIG. 24 is a stress-strain curve of the last cycle, loading profile A.

FIG. 25 is a derivative of the stress-strain curve of the last cycle,loading profile A.

FIG. 26 is a stress-strain curve of the stretch at the first cycle,loading profile B.

FIG. 27 is a derivative of the stress-strain curve of the stretch at thefirst cycle, loading profile B.

FIG. 28A-D are electron transmission micrographs of Sarcophyton softcoral tissue. A. Cluster of cells within the coenenchyme with organelleswhere most probably biosynthesis of the collagen fibers takes place(arrows); B. Higher magnification of the vesicles; C. collagen producingvesicle with fibers; D. Collagen fibers surrounding a sclerite (skeletalelement) of the soft coral (arrows).

FIGS. 29A-D are scanning electron micrographs of Sarcophyton soft coral.A, B. Fibers emerging from the coenenchyme (arrows); C, D. Highmagnification of helical collagen fibers.

FIG. 30A is a general light microscopy image of the collagen of thepresent invention.

FIGS. 30B-C are scanning electron micrographs of a preosteogenic MBA-15cell adhering to the fiber in a resting phase (FIG. 30B and undergoingmitotic division (FIG. 30C).

FIGS. 31A-B are photographs of histological sections of preosteogeniccells grown on the soft coral collagen fibers in vivo. The collagenfibers are marked by (C). New fibrous tissue with areas of higher matrixcontent were formed (circle). No inflammatory cells were visualized.

FIGS. 32A-B are photographs of histological sections of Sarcophyton sp.treated with collagen specific staining Masson Blue. (A) Assemblages offibers stained in blue, located in the mesoglea (the non-cellular partof the soft coral) of polyp mesenteries which run along the polypcavities (×100). (B) Arrangement of the fibers within the mesentery(×400).

FIGS. 33A-B are transmission electron micrographs of collagen fibers ofSarcophyton sp. revealing the fibrils. FIG. 33B (Nikon, 100×10, oilimmersion) is a magnification of FIG. 33A (Nikon, 40×10, oil immersion).

FIG. 34 is a transmission electron micrograph of negatively stainedfibrils that were detached from the fiber by sonication.

FIG. 35 is a transmission electron micrograph of a fibril of the softcoral collagen aligned horizontally by Image-J. The yellow markedrectangular is placed over an area and intensity histogram wasconducted. Image-J averaged the values in the vertical direction at allpoints along the horizontal direction and provided an average intensitydistribution along the collagen fibril. The major bands are at ca 70 nm,but there is some lower amplitude banding inside this 70 nm unit asexpected from negatively stained collagen.

FIG. 36 is a light microscopy image of the fibers according to anembodiment of the present invention (×100, oil immersion).

FIG. 37 is a photograph of a tensometer installed on the dissectingmicroscope for analyzing the collagen fibers isolated from the mesogleaof the soft coral Sarcophyton sp.

FIG. 38 is a graph of a representative stress-strain curve of apreconditioning cycle of the collagen fibers isolated from the mesogleaof the soft coral Sarcophyton sp.

FIG. 39 is a representative stress-strain curve of loading-unloadingcycle (E=0.9 GPa) of the collagen fibers isolated from the mesoglea ofthe soft coral Sarcophyton sp.

FIG. 40 is a representative stress-strain curve of load to failure ofthe collagen fibers isolated from the mesoglea of the soft coralSarcophyton sp.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to acollagen comprising enhanced elasticity and, more particularly, but notexclusively, to a coral-derived collagen.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Collagen is the principal structural protein in the body and constitutesapproximately one-third of the total body protein. It comprises most ofthe organic matter of the skin, tendons, bones and teeth and occurs asfibrous inclusions in most other body structures. Some of the propertiesof collagen are its high tensile strength; its ion exchanging ability,due in part to the binding of electrolytes, metabolites and drugs; itslow antigenicity, due to masking of potential antigenic determinants bythe helical structure, and its low extensibility, semipermeability, andsolubility. Furthermore collagen is a natural substance for celladhesion. These properties make this protein suitable for fabrication ofbioremodelable research products and medical devices such as implantableprostheses, cell growth substrates, and cellular and a-cellular tissueconstructs.

During an underwater excursion on the shores of the Red Sea, the presentinventors identified a coral comprising long fibrous extensions.Following extraction and analysis of these fibers, the present inventorsidentified that they comprise a novel form of collagen. Mechanicaltesting showed that the fibers comprised a unique combination ofproperties being both highly elastic and comprising a high mechanicalstrength (FIGS. 8-10 and 18-27).

Specifically, the coral fibers were shown to have a high reversibleextensibility compared with mammalian collagen fibers (e.g., the coralfibers could be reversibly stretched to strains 2-3 fold greater thanmammalian collagen fibers). Furthermore, the stiffness of the coralfibers was shown to be at the top range of the reported stiffness rangefor mammalian collagen fibers.

Whilst further reducing the present invention to practice, the presentinventors showed that the coral of the present invention could be usedas a scaffolding biomaterial for cell growth and tissue regeneration(FIGS. 30A-C). In vivo subcutaneous transplantation of the fibersrevealed their immunocompetent nature.

Thus, according to one aspect of the present invention, there isprovided an isolated collagen fiber, wherein a length of the fiber priorto stretching by about 15%, is identical to a length of the fiberfollowing the stretching by about 15%.

The term “collagen” as used herein, refers to a polypeptide having atriple helix structure and containing a repeating Gly-X-Y triplet, whereX and Y can be any amino acid but are frequently the imino acids prolineand hydroxyproline. According to one embodiment, the collagen is a typeI, II, III, V, XI, or biologically active fragments therefrom.

As used herein, the phrase “collagen fiber” refers to a non-solubleself-aggregate of the above-mentioned collagen comprising a fibrousstructure in which collagen molecules are packed in series and also inparallel.

According to one embodiment, a length of the fiber prior to stretchingby about 16%, is identical to a length of the fiber following thestretching by about 16%.

According to another embodiment, a length of the fiber prior tostretching by about 17%, is identical to a length of the fiber followingthe stretching by about 17%.

According to another embodiment, a length of the fiber prior tostretching by about 18%, is identical to a length of the fiber followingthe stretching by about 18%.

According to another embodiment, a length of the fiber prior tostretching by about 19%, is identical to a length of the fiber followingthe stretching by about 19%.

According to another embodiment, a length of the fiber prior tostretching by about 20%, is identical to a length of the fiber followingthe stretching by about 20%.

According to another embodiment, a length of the fiber prior tostretching by about 21%, is identical to a length of the fiber followingthe stretching by about 21%.

According to another embodiment, a length of the fiber prior tostretching by about 22%, is identical to a length of the fiber followingthe stretching by about 22%.

According to another embodiment, a length of the fiber prior tostretching by about 23%, is identical to a length of the fiber followingthe stretching by about 23%.

According to one embodiment, the collagen of the present invention doesnot comprise the terminal non-helical regions (telopeptide) existing atboth ends of native collagen—i.e. the collagen comprises atelocollagen.

Contemplated organisms from which the collagen of the present inventionmay be extracted include vertebrate organisms, including mammalianorganisms and fish and invertebrate organisms including cnidaria such asjelly fish and coral.

Exemplary coral from which the collagen of the present invention may beextracted include, but are not limited to Hydrocorals, Stony (Hard)Corals, Colonial Anemones & Button Polyps, Mushroom Coral, Sea Pens,Soft Corals, Gorgonians, Stoloniferans.

According to one embodiment, the coral is the soft coral Sarcophyton sp.

Although the present invention is exemplified by a native collagen (i.e.one that has not been engineered or modified, it will be appreciatedthat the present invention also contemplates genetically modified formsof collagen—for example collagenase resistant collagens and the like [Wuet al., Proc Natl. Acad Sci, Vol. 87, p. 5888-5892, 1990] and otherforms of manufactured collagen.

According to one embodiment, the collagen fiber comprises a NuclearMagnetic Resonance (NMR) spectroscopic profile as shown in FIG. 1.

According to another embodiment, the collagen fiber comprises an aminoacid composition as shown in FIG. 2B.

According to yet another embodiment, a fragment of the collagen fibercomprises a major mass at about 4118.47 mass unit (MU).

According to still another embodiment, the collagen fiber comprises aMass spectroscopy (MS) profile as shown in any one of FIGS. 13A-B-16A-B.

According to yet another embodiment, the isolated collagen fiber of thepresent invention comprises a stiffness (permanent deformation) about30-50% lower than that of mammalian collagen.

Thus, for example, a fiber of about 9 μM in diameter comprises astiffness greater than about 0.34 GPa and less than about 0.54 GPa.According to yet another embodiment, the fiber comprises a stiffnessgreater than about 0.37 GPa. According to yet another embodiment, thefiber comprises a stiffness greater than about 0.4 GPa. According to yetanother embodiment, the fiber comprises a stiffness greater than about0.44 GPa. According to yet another embodiment, the fiber comprises astiffness greater than about 0.5 GPa.

According to yet another embodiment, a bundle of collagen fibers of thepresent invention comprises a stiffness between about 1.5 GPa and 2 GPa.

According to yet another embodiment, the isolated collagen fiber of thepresent invention comprises a tensile strength about half of mammaliancollagen.

Thus, for example a fiber of about 9 μM comprises a tensile strength(breaking point) of more than about 39 MPa. According to yet anotherembodiment, the tensile strength of the fiber is more than about 41 MPa.According to yet another embodiment, the tensile strength of the fiberis more than about 43 MPa. According to yet another embodiment, thetensile strength of the fiber is more than about 45 MPa. According toyet another embodiment, the tensile strength of the fiber is more thanabout 47 MPa. According to yet another embodiment, the tensile strengthof the fiber is more than about 49 MPa. According to yet anotherembodiment, the tensile strength of the fiber is more than about 51 MPa.According to yet another embodiment, the tensile strength of the fiberis more than about 53 MPa. According to yet another embodiment, thetensile strength of the fiber is more than about 55 MPa. According toyet another embodiment, the tensile strength of the fiber is more thanabout 57 MPa. According to yet another embodiment, the tensile strengthof the fiber is more than about 59 MPa. According to yet anotherembodiment, the tensile strength of the fiber is more than about 61 MPa.

It will be appreciated that the collagen fiber of the present inventiontypically comprises three polypeptide chains (alpha chains), wrapped inrope like fashion to form a tight, triple helix structure. According toone embodiment, each alpha chain of the triple helix is separated by thenext by a distance of about 100 nm.

The triple helix of the collagen of the present invention is typicallywound in such a way that peptide bonds linking adjacent amino acids areburied within the interior of the molecule, such that the collagenmolecules are resistant to attack by proteases, such as pepsin.According to one embodiment, the winding is such that the collagen ofthe present invention is resistant to degradation by both trypsin and/orcollagenase.

As used herein, the phrase “resistant to degradation” refers to theinability of the collagen molecule to fully degrade into individualamino acid components.

It will be appreciated that crosslinking may be performed in order toincrease the stability or durability of the collagen. Crosslinking ofcollagen-based materials of the present invention may also be used tosuppress the antigenicity of the material in order to prevent thehyperacute rejection reaction. In addition, crosslinking may used toimprove mechanical properties and enhance resistance to both mechanicaland proteolytic degradation.

Several chemical crosslinking methods for collagen-based materials areknown—see for example U.S. Pat. No. 20050136510. These methods typicallyinvolve the reaction of a bifunctional reagent (i.e., a spacer) with theamine groups of lysine or hydroxylysine residues on differentpolypeptide chains or the activation of carboxyl groups of glutamic andaspartic acid residues followed by the reaction with an amine group ofanother polypeptide chain to give an amide bond. For example,glutaraldehyde (GA), which is a bifunctional aldehyde, or diisocyanatesbridge amine groups on two adjacent polypeptide chains to formcrosslinks. Another method of crosslinking involves the formation of anacyl azide. The acyl azide method involves the activation of carboxylgroups in the polypeptide chain. The activated groups form crosslinks byreaction with collagen amine groups of another chain.

Also, water-soluble carbodiimides can be used to activate the freecarboxyl groups of glutamic and aspartic acid moieties in collagen.Activation of the carboxyl groups with carbodiimides, such as1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.HCl (EDC), givesO-acylisourea groups. A condensation reaction by nucleophilic attack ofa free amine group of a (hydroxy)lysine residue with urea as a leavinggroup results in formation of an amide crosslink. The O-acylisourea canalso be hydrolyzed or rearranged to an N-acylurea, which is much morestable and will not react to form a crosslink. Addition ofN-hydroxysuccinimide (NHS) prevents this rearrangement, however. In thepresence of NHS, the O-acylisourea can be converted to an NHS activatedcarboxyl group, which also can react with a free amine group to form acrosslink.

Other methods of crosslinking may also be used to crosslink the collagenof the present invention such as by glycation using different sugars, byFenton reaction using metal ions such as copper, by lysine oxidaseand/or by UV radiation.

To determine the effect of cross-links and the optimal number ofcross-links per monomer unit, the resilience of a cross-linked polymercan be measured using methods known in the art. The level ofcross-linking can vary provided that the resulting polymer displays therequisite resilient properties. For example, when the cross-linking isby UV-irradiation, the degree of cross-linking is a function of the timeand energy of the irradiation. The time required to achieve a desiredlevel of cross-linking may readily be computed by exposingnon-cross-linked polymer to the source of radiation for different timeintervals and determining the degree of resilience (elastic modulus) ofthe resulting cross-linked material for each time interval. By thisexperimentation, it will be possible to determine the irradiation timerequired to produce a level of resiliency appropriate for a particularapplication. The extent of cross-linking may be monitored during thereaction or pre-determined by using a measured amount of reactants.

The present inventors have shown that the collagen of the presentinvention is bestowed with excellent stretching property and mechanicalstrength without deteriorating a cell adhesion property of collagen.Therefore, application to the uses, where conventional collagen materialcannot be applied due to insufficient stretching property orinsufficient mechanical strength is expected.

The collagen may be used per se, or as part of a composite material. Thecomponents of the composites of the present invention may be attachedto, coated on, embedded or impregnated into the collagen of the presentinvention. In such composites, the collagen may be uncrosslinked,partially crosslined or fully crosslinked. Exemplary components of thecomposite material include, but are not limited to minerals,pharmaceutical agents (i.e. drugs) polysaccharides and polypeptides.

Exemplary polysaccharides that may be used in composite materials of thepresent invention include, but are not limited to glycosaminoglycanssuch as chondroitin sulfate of type A, C, D, or E, dermatan sulfate,keratan sulfate, heparan sulfate, heparin, hyaluronic acid and theirderivatives, individually or mixed.

Exemplary polypeptides that may be used in composite materials of thepresent invention include, but are not limited to silk, elastin andfibronectin.

Exemplary minerals that may be used in composite materials of thepresent invention include, but are not limited to calcium, magnesium,boron, zinc, copper, manganese, iron, silicon, selenium, phosphorus andsulfur. Methods for preparing collagen mineral composites are well knownin the art, see for example WO/2006/118803.

Since the collagen of the present invention has been shown to supportcell propagation, the collagen of the present invention, or compositesthereof may be used as part of a scaffold.

As used herein, the term “scaffold” refers to a 3D matrix upon whichcells may be cultured (i.e., survive and preferably proliferate for apredetermined time period).

The scaffold may be fully comprised of the collagen of the presentinvention or composites thereof, or may comprise a solid support onwhich is layered the collagen of the present invention.

A “solid support,” as used refers to a three-dimensional matrix or aplanar surface (e.g. a cell culture plate) on which cells may becultured. The solid support can be derived from naturally occurringsubstances (i.e., protein based) or synthetic substances. Suitablesynthetic matrices are described in, e.g., U.S. Pat. Nos. 5,041,138,5,512,474, and 6,425,222. For example, biodegradable artificialpolymers, such as polyglycolic acid, polyorthoester, or polyanhydridecan be used for the solid support. Calcium carbonate, aragonite, andporous ceramics (e.g., dense hydroxyapatite ceramic) are also suitablefor use in the solid support. Polymers such as polypropylene,polyethylene glycol, and polystyrene can also be used in the solidsupport.

Therapeutic compounds or agents that modify cellular activity can alsobe incorporated (e.g. attached to, coated on, embedded or impregnated)into the scaffold material or a portion thereof. In addition, agentsthat act to increase cell attachment, cell spreading, cellproliferation, cell differentiation and/or cell migration in thescaffold may also be incorporated into the scaffold. Such agents can bebiological agents such as an amino acid, peptides, polypeptides,proteins, DNA, RNA, lipids and/or proteoglycans.

Suitable proteins which can be used along with the present inventioninclude, but are not limited to, extracellular matrix proteins [e.g.,fibrinogen, collagen, fibronectin, vimentin, microtubule-associatedprotein 1D, Neurite outgrowth factor (NOF), bacterial cellulose (BC),laminin and gelatin], cell adhesion proteins [e.g., integrin,proteoglycan, glycosaminoglycan, laminin, intercellular adhesionmolecule (ICAM) 1, N-CAM, cadherin, tenascin, gicerin, RGD peptide andnerve injury induced protein 2 (ninjurin2)], growth factors [epidermalgrowth factor, transforming growth factor-α, fibroblast growthfactor-acidic, bone morphogenic protein, fibroblast growth factor-basic,erythropoietin, thrombopoietin, hepatocyte growth factor, insulin-likegrowth factor-I, insulin-like growth factor-II, Interferon-β,platelet-derived growth factor, Vascular Endothelial Growth Factor andangiopeptin], cytokines [e.g., M-CSF, IL-1beta, IL-8,beta-thromboglobulin, EMAP-II, G-CSF and IL-10], proteases [pepsin, lowspecificity chymotrypsin, high specificity chymotrypsin, trypsin,carboxypeptidases, aminopeptidases, proline-endopeptidase,Staphylococcus aureus V8 protease, Proteinase K (PK), aspartic protease,serine proteases, metalloproteases, ADAMTS17, tryptase-gamma, andmatriptase-2] and protease substrates.

Additionally and/or alternatively, the scaffolds of the presentinvention may comprise an antiproliferative agent (e.g., rapamycin,paclitaxel, tranilast, Atorvastatin and trapidil), an immunosuppressantdrug (e.g., sirolimus, tacrolimus and Cyclosporine) and/or anon-thrombogenic or anti-adhesive substance (e.g., tissue plasminogenactivator, reteplase, TNK-tPA, glycoprotein IIb/IIIa inhibitors,clopidogrel, aspirin, heparin and low molecular weight heparins such asenoxiparin and dalteparin).

Cells which may be seeded on the collagen of the present invention maycomprise a heterogeneous population of cells or alternatively the cellsmay comprise a homogeneous population of cells. Such cells can be forexample, stem cells (such as embryonic stem cells, bone marrow stemcells, cord blood cells, mesenchymal stem cells, adult tissue stemcells), progenitor cells, or differentiated cells such as chondrocytes,osteoblasts, connective tissue cells (e.g., fibrocytes, fibroblasts andadipose cells), endothelial and epithelial cells. The cells may be naïveor genetically modified.

According to one embodiment of this aspect of the present invention, thecells are mammalian in origin.

Furthermore, the cells may be of autologous origin or non-autologousorigin, such as postpartum-derived cells (as described in U.S.application Ser. Nos. 10/887,012 and 10/887,446). Typically the cellsare selected according to the tissue being generated.

As used herein, the term “seeding” refers to plating, placing and/ordropping cells into the scaffold of the present invention. It will beappreciated that the concentration of cells which are seeded on orwithin the scaffold depends on the type of cells used and thecomposition of the scaffold itself.

Techniques for seeding cells onto or into a scaffold are well known inthe art, and include, without being limited to, static seeding,filtration seeding and centrifugation seeding.

It will be appreciated that to support cell growth, the cells are seededon the collagen of the present invention in the presence of a culturemedium.

The culture medium used by the present invention can be any liquidmedium which allows at least cell survival. Such a culture medium caninclude, for example, salts, sugars, amino acids and minerals in theappropriate concentrations and with various additives and those ofskills in the art are capable of determining a suitable culture mediumto specific cell types. Non-limiting examples of such culture mediuminclude, phosphate buffered saline, DMEM, MEM, RPMI 1640, McCoy's 5Amedium, medium 199 and IMDM (available e.g., from Biological Industries,Beth Ha'emek, Israel; Gibco-Invitrogen Corporation products, GrandIsland, N.Y., USA).

The culture medium may be supplemented with various antibiotics (e.g.,Penicillin and Streptomycin), growth factors or hormones, specific aminoacids (e.g., L-glutamin) cytokines and the like.

The scaffolds of the present invention may be administered to subjectsin need thereof for the regeneration of tissue such as connectivetissue, muscle tissue such as cardiac tissue and pancreatic tissue.Examples of connective tissues include, but are not limited to,cartilage (including, elastic, hyaline, and fibrocartilage), collagen,adipose tissue, reticular connective tissue, embryonic connectivetissues (including mesenchymal connective tissue and mucous connectivetissue), tendons, ligaments, and bone.

Since the scaffolds of the present invention may be used to generatetissue thereon, they may be used for treating subjects with diseasescharacterized by tissue damage or loss.

As used herein, the term “treating” refers to inhibiting or arrestingthe development of a disease, disorder or condition and/or causing thereduction, remission, or regression of a disease, disorder or conditionin an individual suffering from, or diagnosed with, the disease,disorder or condition. Those of skill in the art will be aware ofvarious methodologies and assays which can be used to assess thedevelopment of a disease, disorder or condition, and similarly, variousmethodologies and assays which can be used to assess the reduction,remission or regression of a disease, disorder or condition.

As used herein, the term “subject” refers to mammals, including, but notlimited to, humans, canines and horses.

It will be appreciated that the collagen of the present inventioncomprises a myriad of uses other than for tissue regeneration including,but not limited to treatment of diseases such as interstitial cystitis,scleroderma, and rheumatoid arthritis cosmetic surgery, as a healing aidfor burn patients for reconstruction of bone and a wide variety ofdental, orthopedic and surgical purposes.

As mentioned, the present invention also contemplates biologicallyactive fragments of the collagen of the present invention.

The collagen of the present invention may be formulated aspharmaceutical and/or cosmetic compositions.

The term “cosmetic composition” as used herein refers to a compositionformulated for external application to human or animal skin, nails, orhair for the purpose of beautifying, coloring, conditioning, orprotecting the body surface. The present cosmetic composition can be inany form including for example: a gel, cream, lotion, makeup, coloredcosmetic formulations, shampoo, hair conditioner, cleanser, toner,aftershave, fragrance, nail enamel, and nail treatment product.

The phrase “colored cosmetic formulation” refers to cosmetics containingpigment including for example eye shadow, lipsticks and glosses, lip andeye pencils, mascara, and blush.

As mentioned, the collagen of the present invention may also be used asa cosmetic agent for treatment of skin and hair.

Thus, the present invention contemplates the collagen of the presentinvention as a substance which can be topically applied, optionally incombination with other active substance such as for example a vitamin(vitamin A, C, E or their mixtures) or other topically active substancesincluding but not limited to avarol, avarone or plant extracts, such asExtr. Cepae or Extr. Echinaceae pallidae. The collagen of the presentinvention may be formulated as a topical agent in the form of creams,ointments, lotions or gels such as a hydrogels e.g. on the basis ofpolyacrylate or an oleogel e.g. made of water and Eucerin.

Oleogels comprising both an aqueous and a fatty phase are basedparticularly on Eucerinum anhydricum, a basis of wool wax alcohols andparaffin, wherein the percentage of water and the basis can vary.Furthermore additional lipophilic components for influencing theconsistency can be added, e.g. glycerin, polyethylene glycols ofdifferent chain length, e.g. PEG400, plant oils such as almond oil,liquid paraffin, neutral oil and the like. The hydrogels of the presentinvention can be produced through the use of gel-forming agents andwater, wherein the first are selected especially from natural productssuch as cellulose derivatives, such as cellulose ester and ether, e.g.hydroxyethyl-hydroxypropyl derivatives, e.g. tylose, or also fromsynthetic products such as polyacrylic acid derivatives, such asCarbopol or Carbomer, e.g. P934, P940, P941. They can be produced orpolymerized based on known regulations, from alcoholic suspensions byadding bases for gel formation.

Exemplary amounts of collagen in the gel include 0.01-30 g per 100 g ofgel, 0.01-10 g per 100 g of gel, 0.01-8 g per 100 g of gel, 0.1-5 g per100 g of gel.

The cosmetic composition may comprise other agents capable ofconditioning the body surface including, for example humectants;emollients; oils including for example mineral oil; and shine enhancersincluding for example dimethicone and cyclomethicone. The presentconditioning agents may be included in any of the presentpharmacological and/or cosmetic compositions.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the collagen accountablefor the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, inrtaperitoneal, intranasal, orintraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuosinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (collagen) effective to prevent, alleviate orameliorate symptoms of a disorder (e.g., skin disease).

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to providetissue levels of the active ingredient are sufficient to induce orsuppress the biological effect (minimal effective concentration, MEC).The MEC will vary for each preparation, but can be estimated from invitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as is further detailed above.

Whilst analyzing the coral-derived collagen of the present invention,the present inventors have devised a novel approach to farming thereof.

Thus, according to another aspect of the present invention, there isprovided a method of farming a soft coral, the method comprising:

(a) attaching the soft coral to a clay surface and

(b) growing the soft coral on the clay surface under conditions whichsupport propagation, thereby farming the soft coral.

As used herein, the phrase “soft coral” refers to a coral comprisingpolyps of eight pinnate tentacles. Typically, the soft coral of thepresent invention lack a hard external skeleton.

Exemplary soft corals are provided in Table 1 herein below.

TABLE 1 Common Class/Subclass Order Suborder Family Genus NamesAnthozoa/Octocorallia Alcyonacea Alcyoniina Alcyoniidae Cladiella,Leather Lobophytum, Sinularia, Sarcophyton ″ ″ ″ NephtheidaeDendronephthya, Tree, Nephthea, Cauliflower, Paralemnalia, CarnationScleronephthya ″ ″ ″ Xeniidae Xenia Pulse ″ Helioporacea ″ HelioporidaeHeliopora Blue, Ridge

According to one embodiment the soft coral is of the genus Sarcophyton,such as for example Sarcophyton sp. and Sarcophyton glaucum.

The soft coral may be attached to a clay surface using any method knownin the art, including for example tethering (e.g. plastic ties, rubberbands, wire or thread, stitches, suspension; adhering (e.g.cyanoacrylate/super glue); capturing (e.g. cementing and epoxying); andimpaling (e.g. drilling, pegging and spearing).

The soft coral may be attached to the clay surface immediately followingretrieval from a reef or alternatively may be processed (e.g. bycutting) prior to attachment. According to one embodiment, the softcoral is cut up into pieces of less than about 50 mm² and greater thanabout 25 mm².

Exemplary conditions for propagating the soft coral comprise a watertemperature at a range of about 20-26° C. under a light intensity rangeof about 35-130 μE.

According to one embodiment the pH of the water in which the soft coralis propagated is about 8.2.

According to one embodiment when the temperature is about 20° C., thelight intensity is about 230 μE.

According to one embodiment when the temperature is about 26° C., thelight intensity is about 250 μE.

According to this aspect of the present invention, a soft coral may bepropagated for at least six months, at least one year or even longer. Anincrease of volume of the soft coral cuttings may be as much as X 60following propagation after 8-12 months, according to the method of thepresent invention.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find support inthe following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Protein Analysis of Fibers from the Soft Coral Sarcophyton Sp.Materials and Methods

Extraction of Fibers from the Soft Coral Sarcophyton Sp.:

Samples of the soft coral Sarcophyton sp. were collected from the RedSea and transported frozen to the laboratory. Fibers were mechanicallyremoved from the frozen coral samples.

NMR Analysis:

Collagen fibers ca. 25 mg, were hydrolyzed in 6 N HCl (mL), overnight at110° C. The acid was then removed under vacuum and the residue dissolvedin D2O (0.5 mL). Proton and carbon NMR were measured in 500 MHz and 100MHz NMR machines, respectively. The spectra are characteristic forhydrolyzate of a peptide, namely, a mixture of amino acids.

Amino Acid Analysis:

Waters PicoTag Work Station for gas phase Hydrolysis Hewlet Packard 1090HPLC equipped with Diode array Detector and autoinjector with a PC basedChemstation database, utilizing Amino Quant chemistry was used for theanalysis. In addition, Waters 2690 Alliance HPLC equipped withfluorescence and Diode Array detectors and autoinjector, utilizingAccQ.Tag and or Pico Tag chemistries for the analysis of Hydrolizatesand some physiological Amino acids was used.

Results

Proton and carbon NMR analysis showed that the fibers contain a complexmixture of amino acids, thus indicating that they are composed ofproteins (FIG. 1). Amino acid analysis further confirmed the presence ofamino acids typical to collagen (e.g., high concentration ofGlycine)—FIGS. 2A-B. Enzymes capable of degrading mammalian collagenwere not effective on the collagen of the present invention indicatingthat the latter comprises a different structure to other animalcollagens.

Example 2 Mechanical Characterization of Fibers from the Soft CoralSarcophyton Sp. Materials and Methods

Atomic Force Microscopy:

Atomic force microscopy was performed on a Digital Instruments Dimension3000 atomic force microscope. Images were taken at 256×256 resolutionsin air contact mode. Samples were extracted from mailed specimens withmicro dissecting spring scissors and mounted on freshly-cleaved micawith a protocol similar to that of (Wang et al., 2003, DiabetesMetababolism Research and Reviews 19(4): 288-98; Layton et al., 2004,Journal of Biomechanics 37(6): 879-88).

A total of twelve images were taken as summarized in Table 2.

TABLE 2 Sample number description images taken 010-03-1 purifiedcollagen 5 010-03-2 small fixed sample 4 010-03-3 large unfixed sample 3

Raman Spectroscopy:

Raman Spectroscopy was performed with a Raman RM1000.

Mechanical Testing:

Mechanical testing was performed on the isolated collagen sample. Thesample was taken from the −20° C. storage, and gently dissected with twofine dissecting tweezers so that a single fibrillar portionapproximately 100 μm in diameter and 15 mm long was isolated. The fiberappeared to unwind from the sample in a manner similar to that of abeehive hairdo popular in the United States in the 1960's.

Once the specimen was isolated, it was imaged in brightfield on anOlympus IX81 inverted microscope in air. A total of 25 cross sectionalmeasurements were taken on a total of 17 images. An example of such animage is presented in FIG. 7. The sample was clamped from the bottom ofa Fisher Scientific scientific balance manufactured by DenverInstruments. The device has a maximum load of 1 N and a resolution of 1μN and pulled from the bottom in tension with a micropositioner with aresolution of 10 μm. The specimen was pulled in increments of 10 μm to atotal displacement of 120 μm, or just under 1% strain. At each strainincrement the displacement was held constant for approximately 80seconds. In between displacements, the specimen was allowed to relax forapproximately 20 seconds. During the latter displacements, some stressrelaxation became increasingly evident. At displacements of 100 and 120μm failure events became apparent, perhaps due to sliding amongindividual fibers.

Results

The images obtained following atomic force microscopy are presented inFIGS. 3-5.

FIG. 3 is an image of isolated collagen. A 64-nm spacing was notdetected in these fibrils. A faint hint of ˜100 nm spacing was howeverfound in the fibril marked with a * in FIG. 3. FIGS. 4A-B are imagesfrom small fixed sample illustrating that no fibrillar organization ispresent. FIG. 5 is an image from a large unfixed sample. Probe placementwas difficult and no fibrillar organization was present at the 3 μmscale.

A summary of the peaks found in sample 010-03-1 following Ramanspectroscopy is given in Table 3. These are summarized from Dong, R., etal [Spectrochim Acta A Mol Biomol Spectrosc 60(3): 557-61] and Frushour,B. G., et al [Biopolymers 14(2): 379-91].

TABLE 3 1737 1607 (Phe) 1586 (Pro) 1454 δ(CH3, CH2) 1385 1306 δ(CH2)1166 1122 ν(CCC) 1081 1046  914 ν(C—N) pro  961 Amide III  852 ν(C-C) ofPro ring  835  818 ν(C-C) of backbone or ν(CC)  771  751  657  605  564 542  518

The spectroscopy results are shown in FIG. 6. Peculiar to this sample isthe absence of the peaks at 1670 corresponding to the amide I bonds andthe presence of the peak at 1746 corresponding to the C═O bond.

Mechanical Testing:

Raw force-time data is illustrated in FIG. 8. The raw-force time datawas then converted to force-displacement data by plotting maximum forceattained at each displacement, as illustrated in FIG. 9.

The force displacement curve was then converted to a stress-strain curveaccording to: σ=F/A,

where A is the average initial cross-sectional area calculated accordingto: A=φπd²/4, where φ=0.5 is the approximated volume fraction and d isthe average bundle diameter measured from the optical images obtained.Strain was defined as: ε=Δ/L, where Δ is the displacement and L is theinitial specimen length. A high and a low elastic modulus were alsodetermined based on:E _(LOW)=σ₈₀−σ₁₀/ε₈₀−ε₁₀E _(HIGH)=σ₁₂₀−σ₈₀/ε₁₂₀−ε₈₀

This resulted in an ELOW of 20.6 MPa and an EHIGH of 52.6 MPa.

The stress/strain curve is presented in FIG. 10.

Example 3 MALDI MS Analysis Materials and Methods

Desalting was performed by C18 spin cartridges. Each sample was dilutedto 1 pmol/μl and MALDI MS was performed to analyze the masses present.The samples comprised approximately 100 μg in the first sample and 81.5μg in the second sample.

Results

MALDI MS analysis indicated that the samples comprised one major mass at4118.47 in both samples. FIGS. 11-16 illustrate amino acid normalizationresults for two samples.

Example 4 Comparison of the Mechanical Properties of the Collagen of thePresent Invention and Other Animal Collagens Materials and Methods

Sample Preparation:

Fibers were mechanically removed from frozen coral samples by tweezersand were rolled by an electrical motor to bundles.

Preparing the System:

The sample were frozen (−20° C.) until the day of the experiment. Priorto the experiment, the sample was defrosted for about one hour indistilled water (DW) at room temperature. The sample was weighed,measured and attached at both ends to Perspex clamps, designed to allowfor stretching in the Instron testing machine. During the installation,measures were taken to minimize axial stretching of the sample, and tokeep the sample moist. It must be noted that in some of the samples, thepresence of black nucleolus-like structures were noticed, but they werenot included in the reference length of the sample. The sample wasinstalled in an experimental chamber containing DW, and attached bymeans of the clamps to the Instron instrument (Load cell—5N). The samplewas immersed in a relaxed (un-stretched) state for ca. ½ h.

The Experimental Layout:

Prior to the initiation of the experiment, the undulated sample wasextended until becoming straight but not stretched, and its referencelength was measured. During the experiment, the sample underwent anelongation profile which included N loading cycles. In each cycle thesample was stretched to an elongation level ΔL at a constant rate SR.Later, the stretch was removed at the same rate as illustrated in FIG.17. The loading protocol and the measured load were sampled by theMerlin program (of Instron) at a sampling frequency of 50 Hz.

The Experimental Protocols:

Experiment 1

Data:

Sample number: 4.

Number of fibers in sample (N_(f)): 2750.

Average fiber diameter (d_(f) [μm]): 2.3.

Reference length (L₀ [mm]): 48.

Loading Profiles:

A. Number of cycles (N): 10.

-   -   Strain rate (SR[%/sec]): 0.5.    -   Maximal strain (e₀ [%]): 17.3.

B. Number of cycles (N): 2.

-   -   Strain rate (SR[%/sec]): 0.5.    -   Maximal strain (e₀ [%]): 34.5.

In this experiment, the measurements were conducted using a load cell of100 N. Due to the low magnitude of the measured forces, a largemeasurement noise is expected. According to the information provided,this sample might have disaggregated during its wrapping.

Experiment 2

Data:

Sample number: 1.

Number of fibers in sample (N_(f)): 1800.

Average fiber diameter (d_(f) [μm]): 2.3.

Reference length (L₀ [mm]): 47.

Loading Profiles:

A. Number of cycles (N): 5.

-   -   Strain rate (SR[%/sec]): 0.5.    -   Maximal strain (e₀ [%]): 17.3.

B. Number of cycles (N): 2.

-   -   Strain rate (SR[%/sec]): 0.5.    -   Maximal strain (e₀ [%]): 22.

Results

Results of the experiment include data of time (sec), extension (mm) andload (N). For analyzing the results in a manner which is independent ofthe sample dimensions, the elongation and load were respectivelytransformed to strain (e) and stress (s).

The large deformations axial strain of the sample is given by:e=0.5·(λ²−1)  (1)where λ, the extension ratio, is given by:

$\begin{matrix}{\lambda = {\frac{L}{L_{0}} = {\frac{L_{0} + {extension}}{L_{0}} = {1 + \frac{extension}{L_{0}}}}}} & (2)\end{matrix}$The axial second Piola-Kirchoff stress of the sample is given by:

$\begin{matrix}{s = {\frac{F}{\lambda \cdot A_{0}}\lbrack{Pa}\rbrack}} & (3)\end{matrix}$where F [in Newton] is the load, and −A_(b) [m²] is the cross-sectionalarea of the sample (the fiber bundle) calculated by:

$\begin{matrix}{A_{b} = {{N_{f} \cdot A_{f}} = {N_{f} \cdot {\pi\left( \frac{d_{f}}{2} \right)}^{2}}}} & (4)\end{matrix}$where N_(f) is the number of fibers in a sample and d_(f) is the averagefiber diameter.

Experiment 1:

From observation of the strain-stress curve obtained under loadingprofile A (FIG. 18), the viscoelastic character of the sample can beclearly noted: the hysteresis loop and the decrease in stress values forthe same strain value between consecutive cycles. This reduction instress values became moderate with the increase in the number of cyclesuntil a stable response was obtained. Thus the stable response wasmeasured in the final loading cycle (FIG. 19).

It is clear that the overall response is not a linear one. However, itseems that starting from a certain strain level, there is a linearrelationship between stress and strain. Linear regression to the highrange of strain (17.3%-15.3%) data shows very good correlation(R²=0.999). The estimated slope is 1.765 GPa and is an indication of thestiffness of the sample. In addition, although a correlation analysis atdifferent strain ranges may yield a high R² values, it will becharacterized by a different slope (stiffness) values.

In order to test whether the curve indeed has a linear section, the datawas numerically differentiated. The resulting derivative is presented inFIG. 20.

Numeric differentiation is known to add noise. However, it is possibleto observe that the value of the derivative increases throughout theexperimental stretch range. Hence, from these data it is impossible toconclude that there is indeed a linear relationship between stress andstrain within the strain range used in this protocol.

In order to test the possibility that the linear region occurs at higherstrain levels, the sample must be stretched to higher strains. This goalis achieved by loading profile B.

FIG. 21 presents the stress-strain curve obtained under loading profileB. Three different zones can be identified from the stretching phase ofthe first cycle: 1. A convex non-linear elevation in stress withincreasing strain, 2. A zone of linear stress-strain relationship, 3. Aconcave non-linear stress-strain zone. The thirds zone is most likely aresult of a mechanical failure of some of the fibers. Stretching tohigher strains will eventually lead to failure of all fibers until thestress reduces to zero (tear of the sample). Due to the failure of someof the fibers, the number of active fibers within a sample is unknown(torn fibers will no longer carry load) and thus data from subsequentloading cycles can no longer be directly related to the fibers'properties (since they relate to a sample with fewer fibers). Thereforeonly data from the first loading cycle was used (FIG. 22).

The derivative of the curve is presented in FIG. 23. It appears thatthere is indeed a zone in which the relationship between stress andstrain is linear. Running a linear regression in this zone yields a verygood correlation and a slope (stiffness) of 1.994 GPa.

Experiment 2:

The results of loading profile A are presented in FIGS. 24-25. Similarlyto the previous experiment, a zone of linear stress-strain zone is notapparent. The slope close to the maximal strain used is 1.627 GPa.

Under loading profile B (FIGS. 26-27), three zones can again beidentified in the stress-strain curve. But in contrast to the results ofexperiment #1, it is difficult to conclude with certainty about theexistence of a linear zone from the derivative of the stress-straindata. By running a linear regression near the strain level correspondingto the highest derivative, a slope value of 1.63 GPa is obtained.

Discussion

From the preliminary results presented here, it seems that coral fibershave an impressive stretching ability. The fiber bundle can be stretchedto high strains (17-20%) without failing or undergoing irreversibledamage. According to the literature, mammalian collagen fibers can bereversibly stretched to strains of only about 8-10% without fibers'failure [Fung Y. C., 1993, Biomechanics: Mechanical Properties of LivingTissue, Springer-Verlag, New York, N.Y. pp. 255-260; Sverdlik A., LanirY., 2002, J. Biomech. Eng. Trans. ASME., 124, pp. 78-84].

As opposed to the tendon, a natural source of type 1 collagen, thesample in the present study were artificially prepared by wrapping thefibers into a thick bundle in order to make the stretching load highenough to be reliably measurable. Since the main interest is incharacterizing the properties of a single fiber, the stiffness of thefiber must be estimated from the stiffness of the sample (which wasestimated by data analysis). Therefore the number and diameters offibers within the sample must be determined.

In the tendon, it is known that the collagen fibers are non-uniformlyundulated and that the non-linearity of the stress-strain curve resultsfrom gradual recruitment of the fibers and not necessarily from lack oflinearity of the stress-strain relationship of a single fiber [Fung Y.C., 1993, Biomechanics: Mechanical Properties of Living Tissue,Springer-Verlag, New York, N.Y. pp. 255-260; Sverdlik A., Lanir Y.,2002, J. Biomech. Eng. Trans. ASME., 124, pp. 78-84].

Moreover, since the stress-strain curve is characterized by a linearbehavior at high strain levels, it is likely that a single fiber islinear and that the lack of linearity results only from gradualrecruitment.

From the present results, it appears that there is a zone where a linearrelationship between stress and stain exists.

Assuming this is true, and all fibers are recruited and active, then theoverall stiffness estimate can be used as a measure of the fiberstiffness:

For the first experiment:K _(f) =K _(b)=1.994 GPaFor the second experiment:K _(f) =K _(b)=1.63 GPa

The stiffness data reported in the literature for mammalian collagenfibers are in the range of K_(Collagen)≈0.9÷1.8 GPa [Fung Y. C., 1993,Biomechanics: Mechanical Properties of Living Tissue, Springer-Verlag,New York, N.Y. pp. 255-260; Sverdlik A., Lanir Y., 2002, J. Biomech.Eng. Trans. ASME., 124, pp. 78-84].

Summary

From the above described investigation of the coral fibers, twoprominent findings can be pointed out:

A. The coral fibers have a high reversible extensibility compared withmammalian collagen fibers. (The coral fibers can be reversibly stretchedto strain 2-3 fold greater than collagen fibers).

B. The stiffness of the coral fibers is at the top range of the reportedstiffness range for mammal collagen fibers.

Example 5 Light Microscopy and Electron Microscopy Analysis of theCollagen of the Present Invention Materials and Methods

In order to examine cellular aspects of the soft coral's light, scanningand transmission electron microscopy (SEM, TEM) were applied. Randomsamples were removed from pieces of colonies that were preserved in 2.5%glutaraldehyde in seawater. The samples were decalcified in a mixture ofequal volumes of formic acid (50%) and sodium citrate (15%) for 30minutes and then placed back in 2.5% glutaraldehyde. Samples for lightmicroscopy were placed in Petri dishes (6 cm diameter) and embedded in2% agarose (50° C.) in distilled water. Following its solidification,rectangular pieces closely fitting around each sample were cut out andtransferred to 70% ethanol. This procedure was conducted in order tomaintain the natural orientation of the primary polyps while sectioningthem, thus enabling examination in respect to various parts of thecolony. Following dehydration through a graded series of ethyl alcohol,the samples were embedded in Paraplast (Monoject Scientific) andsections 8 μm thick were cut and stained in hematoxylin and eosin andMasson for visualization of collagen in the tissue. Samples for SEM andTEM were dehydrated through a graded series of ethyl alcohols. Samplesfor SEM were fractured in order to expose their internal parts,critically point dried with liquid CO₂ and then coated with gold.Material was examined under JEOL JSM 840A SEM operated at 25 kV.Material for TEM embedded in Epon and the sections were stained withboth uranyl acetate and lead citrate. The micrographs were studied withJeol 1200 EX electron microscope.

Results

Light microscopy and electron microscopy (SEM and TEM) were performed onthe coral tissue demonstrating fibers in the coenenchyme (mesoglea) ofthe coral arranged as distinct multilayer bundles in the periphery ofthe calcium carbonate skeletal elements (=sclerites) of the coral asillustrated in FIGS. 28A-D. Fiber producing cells were identified andfibers were visualized within intracellular vesicles. SEM micrographsillustrated in FIGS. 29A-D demonstrated the unique helical nature of thefibers.

Example 6 Use of the Fibers of the Present Invention as Scaffold forCell Growth Materials and Methods

Preosteogenic MBA-15 Cells:

Preosteogenic MBA-15 cells were loaded on the collagen fibers of thepresent invention. Cells were cultured in growth medium DulbeccosModified Essential Medium (DMEM) (Gibco, USA) with addition of 10%heat-inactivated fetal calf serum (FCS) (Sigma, USA), 1% glutamine, and1% antibiotics and maintained in 5% CO₂ at 37° C. 5×10⁴ cells/ml wereplated on the polymer and tested after 48 hrs for their interaction withthe substrate.

Scanning Electron Microscopy (SEM) Analysis:

The surfaces of the films with or without cells were observed using aScanning Electron Microscope (SEM, Jeol JEM 6400) at acceleratingvoltage of 5 kV. Sample preparation included fixation in 3%glutaraldehyde (pH 7.4) for 4 hours, immersion in PBS containing 5.4%sucrose for overnight, dehydration with a graded ethanol series, anddrying. The SEM samples were Au/Pd sputtered prior to observation.

Transplantation of Bone Marrow Cells:

Bone marrow cells were loaded on the collagen fibers of the presentinvention. Femur was cleaned from soft tissue and cells were flushed outwith syringe. Cells were allowed to adhere for one hour in vitro andthen were implanted subcutanously in rats.

Results

The prosteogenic cells adhered to the collagen surface and weremaintained on this natural scaffold in vitro as illustrated in FIGS.30A-C.

Bone marrow cells adhered, proliferate and formed new tissue followingtransplantation that allow the evaluation of the fibers as a scaffoldingbiomaterial for cell growth and tissue regeneration. The in vivosubcutaneous transplantation of the fibers revealed theirimmunocompetent nature i.e. no inflammatory reaction was observed in thetissue formed on the scaffold of collagen fibers. As illustrated inFIGS. 31A-B, the tissue formed is fibrous with areas of higher matrixcontent (circle) which indicates deposition, possibly as new osteoid.

In addition, the collagen scaffold alone (without cells) implantedsubcutaneously was adsorbed/degraded within two weeks. When bone marrowcells were added to the collagen scaffold of the present invention andimplanted subcutaneously, fewer osteoclastic cells were observed ascompared to that observed when a commercial collagen scaffold seededwith bone marrow cells was implanted subcutaneously. Further, lesseffective fibro-osteogenic tissue formation was observed with thecommercial collagen scaffold.

Example 7 Propagation of the Soft Coral Genus Sarcophyton (Octocorallia,Alcyonacea)

Species of the soft coral genus Sarcophyton (Octocorallia, Alcyonacea)have a wide Indo-Pacific distribution. They are known for their contentof diverse natural compounds. Sarcophyton glaucum is the most widelydistributed species within the genus, and it is abundant on the Red Seareefs. The present example illustrates the development of a protocol forpropagation of S. glaucum colonies obtained from Eilat (northern RedSea, Israel), in a closed system.

Materials and Methods

The first series of experiments conducted in the study examined: (1) theoptimal size of the cuttings to be used for mass colony formation, (2)the appropriate base type and (3) the best glue to maximize theirsurvival. Cuttings soft coral genus Sarcophyton (Octocorallia,Alcyonacea) were placed in an experimental closed system containing 4 m³of artificial-seawater that fed 24 test tanks (30 l each) enabling aseries of controlled conditions for each experiment. The survival,biomass, organic weight and morphology of the cuttings were monitoredfor 70-80 days of the experiments under different temperature, light,salinity and feeding regimes.

Results

It was found that cuttings with a surface area of 36 mm² polyp-bearingpart (polyparium), glued with Cyanoacrylate glue to clay-made basesyielded the highest survival rate.

At low temperature (20° C.) the highest organic biomass was obtained. Itis suggested that, under these conditions, the calcification rate mightdecrease, thus increasing the organic biomass. Under a high lightintensity of 250 μE, growth rates and dry weight of the cuttingsincreased. Under a mid-range light intensity (35-130 μE) survival andorganic biomass was higher than under the lowest and the highest ones(20 and 250 μE). It is possible that these results were due to lowdensity of the symbiotic zooxanthellae, that under low light intensitiesdo not allow efficient photosynthesis, and to photoinhibition takingplace under high light intensities.

Salinity did not affect survival, biomass and organic weight of thecuttings over time.

Cuttings that were frequently fed by brine shrimps-Artemia nauplei(every 2 days) had the lowest organic weight. It is suggested that thetime required for digestion by the polyps may control the rate of foodcapture and, therefore, frequent feeding will not benefit the cuttingsand may even harm them, due to the decomposition of unconsumed food inthe water that impairs the water quality.

Observations derived from the experiments indicated that the volume ofwater in which the cuttings are reared may influence their ability toadhere to the substrate. Experiments showed that cuttings maintained ina small water volume (20 or 70 l.) attached to the clay bases fasterthan cuttings kept in 2,880 liter aquaria.

In order to examine the relationship between colony size and percentageof organic matter, reefal colonies of S. glaucum, of three size classes(5-7 cm, 10-15 cm, 20 cm disc diameter) were examined. The lowestpercentage of organic matter (10%) was found in the small colonies,which were also used in the current study for preparation of thecuttings. The percentage of organic matter in cuttings reared in theclosed system was noticeably higher than that found in all the threesize groups collected from the sea. It is possible that reefal colonieshave more calcareous material in the coral sclerites, facilitating theiradaptation to water currents, waves and predation.

In a concluding experiment cuttings were reared under a culturingprotocol based on the results of the above-mentioned experiments. Theresults were compared with those obtained in an open seawater system andin the reef. It was concluded that a closed system increased thepercentage of organic matter of the cuttings. Although cuttings thatwere farmed in the sea had a slightly higher dry weight compared tothose farmed in closed system, the former had a much higher percentageof organic matter, which is extremely important for the pharmaceuticalindustry. Cuttings from the closed system had a higher dry weight andorganic matter percentage compared to those farmed in the open system.

Example 8 Composition of the Soft Coral Genus Sarcophyton glaucum(Octocorallia, Alcyonacea) Following Farming in a Closed System Results

NMR (Nuclear Magnetic Resonance) analysis revealed different patterns ofnatural compounds in cuttings reared in the three different environmentsmentioned above (i.e., closed seawater system, open seawater system andon the reef). Since all cuttings were obtained from the same parentcolonies, it is suggested that the environmental conditions of theexperimental set up determined the content of the natural compounds.

Cuttings reared in the sea were characterized by a semi-solid rigidityand had a hemispheric polyparium and short polyps. Cuttings from theclosed system had long polyps, a long colony stalk and a flatpolyparium.

Conclusion

The results of the experiments described in Examples 7 and 8, illustratethe advantages of a closed system for culturing of S. glaucum.

Example 9 Structural Features of the Soft Coral Collagen Fibers

In order to analyze the structural features of the soft coral collagenfibers of one embodiment of the present invention, histological sectionsof Sarcophyton sp. were treated with collagen specific staining MassonBlue. In addition, the collagen fibers of one embodiment of the presentinvention were analyzed using transmission electron microscopy (TEM).

Results

The results of the histological staining of the fibers are provided inFIGS. 32A-B.

The TEM images are provided in FIGS. 33A-B, 34 and 35. In the images thegreen is collagen fibers and the red is cytoplasm of the soft coralcells.

Example 10 Mechanical Properties of Isolated Collagen Fibers fromSarcophyton Sp Materials and Methods

Sample Preparation:

Bundles of fibers were isolated from the mesoglea of the soft coralSarcophyton sp. by forcipes, and starched on polyethylene plastic cards(1×8 cm) prior to incubation in ethanol (70% in fresh water). Thesamples were then analyzed.

Experimental System:

Prior to the experiment, samples were rehydrated for one hour in freshwater (FW) at room temperature. Single fibers were isolated from thebundles by forcipes under a dissecting microscope and measured (lengthand diameter) by light microscope (Nikon, 100×, oil immersion)photography and imaging software (FIG. 36). A single fiber was attachedat one end to a stainless steel tensometer beam with a half bridgeformed by two semi-conductor strain gages (1 gr=7.45V, max 10 gr) usingcynoacrylate glue. The other end of the fiber was fixed to a stainlesssteel beam maneuvered by micrometer (FIG. 37). During installation,measures were taken to minimize axial stretching of the sample and tokeep the sample moist. The sample was installed in an experimentalchamber containing FW, and was immersed in a relaxed (un-stretched)state for approximately 30 minutes prior to testing.

Before initiating the experiments, the force transducer beam deflectionwas measured by pulling a thin, non-flexible copper wire in knownincrements. The calculated beam deflection was subtracted from all fiberdisplacement results in order to obtain the true displacement. In theexperiments, the undulated sample was extended until it became straightbut not stretched, and its reference length was measured by caliber inmm (L initial). Preconditioning to 5% (3 cycles) was performed beforethe samples underwent elongation profiles of load-unload (4 cycles) orLoad to failure, and L0 was measured. Volts output was gained ×1000 andreading was performed by a voltmeter. Micrometer maneuvering and datarecording was performed by hand in 100 μM increments and Force (N) wascalculated.

Results of the experiment include extension (μm) and load (N). Foranalyzing the results in a manner that is independent of the sampledimensions, the elongation and load were respectively transformed tostrain (e) and stress (s).

s=F/A; e=ΔL/L0, where F [in Newton] is the load, and A=πR² is thecross-sectional area of the sample (the fiber). Data analysis wasperformed using Microsoft Excel.

Results

Preconditioning:

L0 was obtained from strain-stress curves of preconditioning cycles(FIG. 38).

Average different between L initial and L0 was 0.03±0.0094.

Load-Unload Cycles:

From observation of the strain-stress curves obtained underloading-unloading cycles of 3 samples, the viscoelastic character of thesamples can be observed: the hysteresis loop and the decrease in stressvalues for the same strain value between consecutive cycles (FIG. 39).Average hysteresis for the first cycle was 41.2595±15.5%.

It is clear from FIG. 39 that the overall response is not a linear one.However, it seems that at a certain strain level, there is a linearrelationship between stress and strain. Linear regression to the highrange of strain (7%-15%), shows very good correlation (R²=0.999). Nocorrelation was found between slope to sample length (p>0.05). Theestimated slope is 0.5±0.1 GPa and is an indication of the stiffness ofthe sample.

Load to Failure:

From the load to failure strain-stress curves of 12 samples, it is clearthat the overall response is not a linear one (FIG. 40). Linearregression to the high range of strain (8%-19.4%) shows very goodcorrelation (R²=0.999). No correlation was found between slope to samplelength (p>0.05). The estimated slope is 0.44±0.1 GPa and is anindication of the stiffness of the sample. Average load to failure was49.4±11.7 MPa and average extensibility was 19.4±4.27%.

Conclusion

From the results presented here, it seems that soft coral fibers have animpressive stretching ability. The fibers can be stretched to highstrains (19.4±4.27%) without failing or undergoing irreversible damage.According to the literature, mammalian collagen fibers can be reversiblystretched to strains of only about 8-10% without fibers' failure (FIG.40) (Fung, 1993, Biomechanics: Mechanical Properties of Living Tissue,Springer-Verlag, New York, N.Y. pp. 255-260 which is hereby incorporatedby reference) and mesoglea can be stretched to strains of 3.5-6% (Koehl,1982, Mechanical design of spicule-reinforced connective tissue:stiffness. J. Exp. Biol. 98, 239-267). The stiffness of the coral fibers(0.44±0.1 GPa) is about half to a third lower than reported stiffnessrange for mammalian collagen fibers (0.9-1.8 GPa) (Sverdlik & Lanir.,2002, J. Biomech. Eng. Trans. ASME., 124, pp. 78-84), and five orders ofmagnitude bigger then mesoglea (0.01 MPa) (Vogel, 2003, ComparativeBiomechanics: Life's Physical World. Princeton: Princeton UniversityPress). Their average load to failure (49.4±11.7 MPa) is about a half ofthe reported tensile strength for mammalian collagen fibers (100 MPa)(Vogel, 2003, supra) and an order of magnitude bigger than mesoglea(1-2.5 MPa) (Koehl, 1981, supra).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A method of regenerating connective tissue, themethod comprising providing to a subject in need-thereof a scaffoldcomprising collagen fibers being extracted from a soft coral, wherein alength of said fibers following stretching by about 15%, is identical toa length of said fibers prior to said stretching, thereby regeneratingthe connective tissue.
 2. The method of claim 1, wherein the collagen iscomprised in a composite.
 3. The method of claim 2, wherein a secondcomponent of said composite is selected from the group consisting of amineral, a polysaccharide and a polypeptide.
 4. The method of claim 1,wherein said connective tissue is selected from the group consisting ofcartilage, collagen, adipose tissue, reticular connective tissue,embryonic connective tissues, tendons, ligaments, and bone.
 5. Themethod of claim 4, wherein said cartilage is selected from the groupconsisting of elastic cartilage, hyaline cartilage and fibrocartilage.6. The method of claim 1, wherein said soft coral is of the Sarcophytongenus.
 7. The method of claim 1, wherein said collagen fibers have astiffness about 30-50% lower than that of mammalian collagen.
 8. Themethod of claim 1, wherein said collagen fibers have a tensile strengthbetween about 39-59 MPa.
 9. A method of regenerating connective tissue,the method comprising providing to a subject in need-thereof a scaffoldcomprising collagen fibers being extracted from a soft coral of theSarcophyton genus.
 10. The method of claim 9, further comprising seedingcells on the scaffold prior to said providing.